Maria Hägg Olofsson

Inhibition of proteasome deubiquitinating activity as a new cancer therapy

Ubiquitin-tagged substrates are degraded by the 26S proteasome, which is a multisubunit complex comprising a proteolytic 20S core particle capped by 19S regulatory particles. The approval of bortezomib for the treatment of multiple myeloma validated the 20S core particle as an anticancer drug target. Here we describe the small molecule b-AP15 as a previously unidentified class of proteasome inhibitor that abrogates the deubiquitinating activity of the 19S regulatory particle. b-AP15 inhibited the activity of two 19S regulatory-particle–associated deubiquitinases, ubiquitin C-terminal hydrolase 5 (UCHL5) and ubiquitin-specific peptidase 14 (USP14), resulting in accumulation of polyubiquitin. b-AP15 induced tumor cell apoptosis that was insensitive to TP53 status and overexpression of the apoptosis inhibitor BCL2. We show that treatment with b-AP15 inhibited tumor progression in four different in vivo solid tumor models and inhibited organ infiltration in an acute myeloid leukemia model. Our results show that the deubiquitinating activity of the 19S regulatory particle is a new anticancer drug target.

Cytokeratin-18 is a useful serum biomarker for early determination of response of breast carcinomas to chemotherapy.

Purpose: With a widening arsenal of cancer therapies available, it is important to develop therapy-specific predictive markers and methods to rapidly assess treatment efficacy. We here evaluated the use of cytokeratin-18 (CK18) as a serum biomarker for monitoring chemotherapy-induced cell death in breast cancer. Experimental Design: Different molecular forms of CK18 (caspase cleaved and total) were assessed by specific ELISA assays. Drug-induced release of CK18 was examined from breast carcinoma cells and tissue. CK18 protein composition was examined in serum. CK18 levels were determined in serum from 61 breast cancer patients during docetaxel or cyclophosphamide/epirubicin/5-fluorouracil (CEF) therapy. Results: Caspase-cleaved CK18 molecules were released from monolayer cultures and tumor organ cultures to the extracellular compartment. CK18 was present in complexes with other cytokeratins in serum. Such CK18 protein complexes are remarkably stable, leading to favorable performance of CK18 biomarker assays for clinical investigations. Docetaxel induced increased levels of caspase-cleaved CK18 in serum from breast cancer patients, indicating apoptosis. CEF therapy led to increases predominantly in uncleaved CK18, indicating induction of necrotic cell death in many tumors. The increase in total CK18 at 24 h of the first treatment cycle correlated to the clinical response to CEF therapy (P < 0.0001). Conclusions: Induction of necrotic cell death may explain the clinical efficacy of anthracycline-based therapy for breast carcinomas with defective apoptosis pathways. We suggest that CK18 biomarkers are useful for early prediction of the response to CEF therapy in breast cancer and may be useful biomarkers for clinical trials.

Induction of mitochondrial dysfunction as a strategy for targeting tumour cells in metabolically compromised microenvironments.

Abnormal vascularization of solid tumours results in the development of microenvironments deprived of oxygen and nutrients that harbour slowly growing and metabolically stressed cells. Such cells display enhanced resistance to standard chemotherapeutic agents and repopulate tumours after therapy. Here we identify the small molecule VLX600 as a drug that is preferentially active against quiescent cells in colon cancer 3-D microtissues. The anticancer activity is associated with reduced mitochondrial respiration, leading to bioenergetic catastrophe and tumour cell death. VLX600 shows enhanced cytotoxic activity under conditions of nutrient starvation. Importantly, VLX600 displays tumour growth inhibition in vivo. Our findings suggest that tumour cells in metabolically compromised microenvironments have a limited ability to respond to decreased mitochondrial function, and suggest a strategy for targeting the quiescent populations of tumour cells for improved cancer treatment.

Utilization of cytokeratin- based biomarkers for pharmacodynamic studies

Cytokeratin (CK)18 is a useful serum biomarker for the determination of cell death of epithelial-derived tumors (carcinomas). ELISAs are available for caspase-cleaved CK18 (M30) released from apoptotic cells, or total CK18 (M65) released by cells undergoing cell death by any cause. These assays have been demonstrated to have prognostic or predictive utility in various types of carcinomas. Encouraging data have been reported by different investigators with regard to the potential use of CK18 as a serum efficacy biomarker for monitoring therapy efficiency in carcinoma patients. The ratio of caspase-cleaved to total CK18 can be determined conveniently in serum or plasma using commercially available ELISA kits (M30-Apoptosense® and M65® ELISA, Peviva AB, Bromma, Sweden). M30:M65 ratios potentially provide information as to whether tumor cells undergo apoptosis or necrosis. However, as discussed in this review, M30:M65 ratios should be interpreted with caution and, preferably, only be applied to samples that contain significant levels of CK18. We conclude that M30 and M65 biomarkers provide both quantitative and qualitative information on carcinoma cell death.

Three-Dimensional Cell Culture-Based Screening Identifies the Anthelmintic Drug Nitazoxanide as a Candidate for Treatment of Colorectal Cancer

Because dormant cancer cells in hypoxic and nutrient-deprived regions of solid tumors provide a major obstacle to treatment, compounds targeting those cells might have clinical benefits. Here, we describe a high-throughput drug screening approach, using glucose-deprived multicellular tumor spheroids (MCTS) with inner hypoxia, to identify compounds that specifically target this cell population. We used a concept of drug repositioning—using known molecules for new indications. This is a promising strategy to identify molecules for rapid clinical advancement. By screening 1,600 compounds with documented clinical history, we aimed to identify candidates with unforeseen potential for repositioning as anticancer drugs. Our screen identified five molecules with pronounced MCTS-selective activity: nitazoxanide, niclosamide, closantel, pyrvinium pamoate, and salinomycin. Herein, we show that all five compounds inhibit mitochondrial respiration. This suggests that cancer cells in low glucose concentrations depend on oxidative phosphorylation rather than solely glycolysis. Importantly, continuous exposure to the compounds was required to achieve effective treatment. Nitazoxanide, an FDA-approved antiprotozoal drug with excellent pharmacokinetic and safety profile, is the only molecule among the screening hits that reaches high plasma concentrations persisting for up to a few hours after single oral dose. Nitazoxanide activated the AMPK pathway and downregulated c-Myc, mTOR, and Wnt signaling at clinically achievable concentrations. Nitazoxanide combined with the cytotoxic drug irinotecan showed anticancer activity in vivo. We here report that the FDA-approved anthelmintic drug nitazoxanide could be a potential candidate for advancement into cancer clinical trials. Mol Cancer Ther; 14(6); 1504–16. ©2015 AACR.

Identification of a Novel Topoisomerase Inhibitor Effective in Cells Overexpressing Drug Efflux Transporters

Background Natural product structures have high chemical diversity and are attractive as lead structures for discovery of new drugs. One of the disease areas where natural products are most frequently used as therapeutics is oncology. Method and Findings A library of natural products (NCI Natural Product set) was screened for compounds that induce apoptosis of HCT116 colon carcinoma cells using an assay that measures an endogenous caspase-cleavage product. One of the apoptosis-inducing compounds identified in the screen was thaspine (taspine), an alkaloid from the South American tree Croton lechleri. The cortex of this tree is used for medicinal purposes by tribes in the Amazonas basin. Thaspine was found to induce conformational activation of the pro-apoptotic proteins Bak and Bax, mitochondrial cytochrome c release and mitochondrial membrane permeabilization in HCT116 cells. Analysis of the gene expression signature of thaspine-treated cells suggested that thaspine is a topoisomerase inhibitor. Inhibition of both topoisomerase I and II was observed using in vitro assays, and thaspine was found to have a reduced cytotoxic effect on a cell line with a mutated topoisomerase II enzyme. Interestingly, in contrast to the topoisomerase II inhibitors doxorubicin, etoposide and mitoxantrone, thaspine was cytotoxic to cell lines overexpressing the PgP or MRP drug efflux transporters. We finally show that thaspine induces wide-spread apoptosis in colon carcinoma multicellular spheroids and that apoptosis is induced in two xenograft mouse models in vivo. Conclusions The alkaloid thaspine from the cortex of Croton lechleri is a dual topoisomerase inhibitor effective in cells overexpressing drug efflux transporters and induces wide-spread apoptosis in multicellular spheroids.

The proteasome deubiquitinase inhibitor VLX1570 shows selectivity for ubiquitin-specific protease-14 and induces apoptosis of multiple myeloma cells

Inhibition of deubiquitinase (DUB) activity is a promising strategy for cancer therapy. VLX1570 is an inhibitor of proteasome DUB activity currently in clinical trials for relapsed multiple myeloma. Here we show that VLX1570 binds to and inhibits the activity of ubiquitin-specific protease-14 (USP14) in vitro, with comparatively weaker inhibitory activity towards UCHL5 (ubiquitin-C-terminal hydrolase-5). Exposure of multiple myeloma cells to VLX1570 resulted in thermostabilization of USP14 at therapeutically relevant concentrations. Transient knockdown of USP14 or UCHL5 expression by electroporation of siRNA reduced the viability of multiple myeloma cells. Treatment of multiple myeloma cells with VLX1570 induced the accumulation of proteasome-bound high molecular weight polyubiquitin conjugates and an apoptotic response. Sensitivity to VLX1570 was moderately affected by altered drug uptake, but was unaffected by overexpression of BCL2-family proteins or inhibitors of caspase activity. Finally, treatment with VLX1570 was found to lead to extended survival in xenograft models of multiple myeloma. Our findings demonstrate promising antiproliferative activity of VLX1570 in multiple myeloma, primarily associated with inhibition of USP14 activity.

Identification of Agents that Induce Apoptosis of Multicellular Tumour Spheroids: Enrichment for Mitotic Inhibitors with Hydrophobic Properties

Cell‐based anticancer drug screening generally utilizes rapidly proliferating tumour cells grown as monolayer cultures. Hit compounds from such screens are not necessarily effective on hypoxic and slowly proliferating cells in 3‐D tumour tissue. The aim of this study was to examine the potential usefulness of 3‐D cultured tumour cells for anticancer drug screening. We used colon carcinoma multicellular spheroids containing hypoxic and quiescent cells in core areas for this purpose. Three libraries (∼11 000 compounds) were screened using antiproliferative activity and/or apoptosis as end‐points. Screening of monolayer and spheroid cultures was found to identify different sets of hit compounds. Spheroid screening enriched for hydrophobic compounds: median XLogP values of 4.3 and 4.4 were observed for the hits in two independent screening campaigns. Mechanistic analysis revealed that the majority of spheroid screening hits were microtubuli inhibitors. One of these inhibitors was examined in detail and found to be effective against non‐dividing cells in the hypoxic centres of spheroids. Spheroid screening represents a conceptually new strategy for anticancer drug discovery. Our findings have implications for drug library design and hit selection in projects aimed to develop drugs for the treatment of solid tumours.

Restriction of cisplatin induction of acute apoptosis to a subpopulation of cells in a three-dimensional carcinoma culture model

Cisplatin is a clinically important chemotherapeutical agent used to treat epithelial malignancies. High concentrations (20–100 μM) of cisplatin have been used in numerous studies to induce apoptosis of carcinoma cells grown in monolayer culture over 24–48 hr. These conditions may not be relevant to 3‐D tumor tissue in vivo and the importance of apoptosis for tumor response is controversial. We here studied the effects of cisplatin on a 3‐D colon carcinoma in vitro model (multicellular spheroids). Cisplatin at a dose of 40 μM induced active caspase‐3 preferentially in the peripheral 30 μm cell layer of spheroids, mainly during late stages (72–96 hr). The p53 response to cisplatin was also largely confined to peripheral cell layers. Despite the use of a high cisplatin concentration, a significant fraction of the cells in the spheroids survived treatment. A high proportion of surviving cells stained positive for β‐galactosidase, a marker of premature senescence. Cells growth‐arrested by cisplatin treatment showed a higher spontaneous cell death rate than untreated proliferating cells. We propose that acute apoptosis is of minor significance for the overall response of carcinoma cells to cisplatin treatment.

HER2-Positive Tumors Imaged Within 1 Hour Using a Site-Specifically 11C-Labeled Sel-Tagged Affibody Molecule

A rapid, reliable method for distinguishing tumors or metastases that overexpress human epidermal growth factor receptor 2 (HER2) from those that do not is highly desired for individualizing therapy and predicting prognoses. In vivo imaging methods are available but not yet in clinical practice; new methodologies improving speed, sensitivity, and specificity are required. Methods: A HER2-binding Affibody molecule, ZHER2:342, was recombinantly fused with a C-terminal selenocysteine-containing tetrapeptide Sel-tag, allowing site-specific labeling with either 11C or 68Ga, followed by biodistribution studies with small-animal PET. Dosimetry data for the 2 radiotracers were compared. Imaging of HER2-expressing human tumor xenografts was performed using the 11C-labeled Affibody molecule. Results: Both the 11C- and 68Ga-labeled tracers initially cleared rapidly from the blood, followed by a slower decrease to 4–5 percentage injected dose per gram of tissue at 1 h. Final retention in the kidneys was much lower (>5-fold) for the 11C-labeled protein, and its overall absorbed dose was considerably lower. 11C-ZHER2:342 showed excellent tumor-targeting capability, with almost 10 percentage injected dose per gram of tissue in HER2-expressing tumors within 1 h. Specificity was demonstrated by preblocking binding sites with excess ligand, yielding significantly reduced radiotracer uptake (P = 0.002), comparable to uptake in tumors with low HER2 expression. Conclusion: To our knowledge, the Sel-tagging technique is the first that enables site-specific 11C-radiolabeling of proteins. Here we present the finding that, in a favorable combination between radionuclide half-life and in vivo pharmacokinetics of the Affibody molecules, 11C-labeled Sel-tagged ZHER2:342 can successfully be used for rapid and repeated PET studies of HER2 expression in tumors.